PROJECT SUMMARY/ABSTRACT Mineralized enamel is the hardest tissue in the human body, and its proper formation is critical for the protection and maintenance of healthy teeth for a lifetime. Enamel biomineralization is directed by the epithelial derived ameloblasts. Ameloblasts secrete enamel matrix proteins, which are then hydrolyzed and replaced by hydroxyapatite crystals. Maturation stage ameloblasts modulate in waves to acidify approximately 80% of the mineralizing maturation stage matrix, with the remaining approximately 20% of the matrix remaining neutral. When pH cycling is disrupted, enamel formation is dysregulated, and the resulting enamel matrix is hypomineralized. Though an acidic environment is unfavorable to biomineralization, matrix acidification in enamel formation has been presumed to have a role in refinement of the hydroxyapatite (HAP) enamel crystals. However the mechanism(s) that control pH cycling remain unclear. In this project, we will use our novel polarized ameloblast culture system, along with Wdr72-/- and Cftr-/- mouse models, to test our central hypothesis that ameloblasts directly regulate pH cycling in the enamel matrix, to optimize enamel matrix mineralization. We will test this central hypothesis with the following specific aims. 1) To determine the role of ameloblasts in acidifying the enamel matrix; 2) To determine the effects of extracellular pH on calcium transport by ameloblasts; 3). To determine the role of matrix acidification on protein hydrolysis and HAP crystal formation. These studies will allow us to better understand the etiology of enamel hypomineralization, and will allow us to apply this knowledge to reduce the risk for of enamel defects. The long-term goal of our research is to determine how pH cycling by ameloblasts directs enamel matrix biomineralization to synthesize the unique prismatic mineralized structure that forms tooth enamel.